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Cell-Free Protein Expression

Proteins were expressed using the TNT® SP6 High-Yield Wheat Germ Protein Expression System (Cat.# L3260). In a typical reaction, 10μg of vector was added to 30μl of lysate, then water was added to a final reaction volume of 50μl. The reaction was incubated at 25°C for two hours with mixing (1400rpm max speed) on a thermomixer (Eppendorf).

DNA Probes

DNA probes were synthesized and purified by Integrated DNA Technologies. The two complementary strands were annealed prior to protein binding. The sense strand contained a 5´Alexa647 fluorophore. The WT-DNA (5´Alexa647-AGTTGAGGGGACTTTCCCAGGC´3) contained the Ig-κB dimer target site, while the Mut-DNA (5´Alexa647-AGTTGAGGTGTGTGTGTCAGGC) had the Ig-κB dimer target site replaced with (GT)5.

DNA:Protein Binding Experiments

The HaloLink™ Protein Array System was used to analyze DNA:protein interactions. Slides were assembled according to the manufacturer’s protocol. The HaloTag® Standard Protein (5μl of a serial dilution) in 1X PBSB (1X PBS + 10mg/ml of BSA) was added at concentrations of 0, 1.3, 2.6, 5.2, 10.5, 21, 42, 83, 166 or 332nM to wells 1–10 of the first column of wells (Column A). This was used as the protein concentration standard on the slide. Lysate (5μl) expressing a HaloTag® fusion protein was added to wells of the slide. Typically, three wells are dedicated to determining protein concentration. The HaloTag® fusion proteins were incubated at room temperature for one hour in a humidity chamber (see manufacturer’s protocol for instructions for making chamber). After the incubation, the slides were washed with 1X PBSI (1X PBS + 0.05% IGEPAL® CA-630) using a gentle stream from a squirt bottle. The slide was immediately centrifuged at 350 × g in a 50ml conical tube. To determine the protein concentration, 5μl of a 1:200-fold dilution of Anti-HaloTag® pAb (10μg/ml) was added to the appropriate wells (including Column A). After a one-hour incubation at room temperature in a humidity chamber, the slide was washed and centrifuged at 350 × g in a 50ml conical tube. Next, 5μl of a 1:200-fold dilution of Alexa® Fluor 647 anti-rabbit IgG antibody (10μg/ml) (Invitrogen) was added to the wells that had antibody added. To analyze the DNA:protein interactions, 5μ of DNA probe (at various concentrations) was added to each well. After a one-hour incubation at room temperature in a humidity chamber, the slide was washed and centrifuged at 350 × g in a 50ml conical tube. The silicon gasket was removed, and the slide was analyzed with a fluorescent slide reader. For these experiments, we scanned with a GenePix® slide scanner (Molecular Devices) at a wavelength of 635nm.

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